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Refolding active human DNA polymerase nu from inclusion bodies
Mercedes E Arana1, Gary K Powell, Lori L Edwards
1Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Protein Expression and Purification
|October 27, 2009
Summary
High hydrostatic pressure successfully refolded insoluble human DNA polymerase nu (Pol nu) from inclusion bodies into a soluble and active form. This method may aid in studying other difficult-to-purify DNA polymerases.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Human DNA polymerase nu (Pol nu) is a family A DNA polymerase with an unclear biological role.
- Overexpression in Escherichia coli results in insoluble Pol nu, hindering purification and functional studies.
Purpose of the Study:
- To develop a method for obtaining soluble and active human Pol nu for biochemical characterization.
- To investigate the potential of high hydrostatic pressure for refolding insoluble proteins.
Main Methods:
- Overexpression of human Pol nu in Escherichia coli.
- Inclusion body isolation and solubilization.
- Refolding of Pol nu using high hydrostatic pressure treatment.
- Biochemical and physical characterization of refolded Pol nu.
Main Results:
- High hydrostatic pressure effectively refolded Pol nu from inclusion bodies into a soluble and active state.
- The refolded Pol nu exhibited properties comparable to the small amount of naturally soluble Pol nu.
- This pressure-induced refolding strategy yielded functionally active enzyme.
Conclusions:
- High hydrostatic pressure is a viable method for refolding insoluble human Pol nu.
- This technique can enable the biochemical and functional characterization of Pol nu.
- The approach may be broadly applicable to other overexpressed, insoluble DNA polymerases.
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